Compositions and methods for inhibiting seizures

ABSTRACT

Provided herein are methods and compositions related to treating or preventing seizures. In some aspects, provided herein are methods of treating or preventing seizures in a subject by administering to the subject a composition comprising Parabacteroides and Akkermansia bacteria.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/436,711, filed Dec. 20, 2016, and U.S.Provisional Patent Application Ser. No. 62/447,992 filed Jan. 19, 2017,each of which are herein incorporated by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant NumberGM065823 and Grant Number GM106996, awarded by the National Institutesof Health. The Government has certain rights in the invention.

BACKGROUND

Epilepsy is characterized by recurrent seizures that can lead to loss ofawareness, loss of consciousness, and/or disturbances of movement,autonomic function, sensation (including vision, hearing and taste),mood, and/or mental function. Epilepsy afflicts 1-2% of the populationin the developed world.

The low-carbohydrate, high-fat ketogenic diet (KD) is a treatment forrefractory epilepsy, wherein more than one-third of epilepticindividuals do not respond to existing anticonvulsant medications. Theefficacy of the KD is supported by multiple retrospective andprospective studies, which estimate that ˜30% of patients becomeseizure-free, and ˜60% experience significant benefit. However, despiteits value for treating epilepsy and its increasing application to otherdisorders, including autism, Alzheimer's disease, Parkinson's disease,metabolic syndrome and cancer, use of the KD remains low due todifficulties with implementation, dietary compliance and adverse sideeffects. In fact, even with successful seizure reduction, epilepticpatient retention on the KD is only an estimated 12% by the third yearof dietary therapy. Moreover, mechanisms underlying the beneficialeffects of the KD are poorly understood, and molecular and/or cellulartargets for intervention are lacking. That the diet succeeds incontrolling various types of symptoms in cases when drugs fail suggeststhat it enhances endogenous neuroprotective pathways that are nottargeted by existing medications.

SUMMARY

Provided herein are methods and compositions for mimicking the effectsof a ketogenic diet by administering probiotic compositions to asubject. In certain embodiments, the methods and compositions are forthe treatment or prevention of seizures in a subject (e.g., a subjectwith a neurodevelopmental disorder, such as autism spectrum disorder,Rett syndrome, fragile X, attention deficit disorder (ADD), attentiondeficit/hyperactivity disorder (ADHD), refractory epilepsy, and/ornon-refractory epilepsy). In other embodiments, the methods andcompositions are for preventing or treating a condition (e.g., epilepsy,seizures, autism spectrum disorder, Alzheimer's disease, Huntington'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),cancer, stroke, a metabolic disease (e.g., diabetes or obesity), amitochondrial disorder, depression, migraines (e.g., chronic migraines),Rett syndrome, attention deficit disorder, fragile X syndrome, ortraumatic brain injury (TBI)) in a subject. Preferably, the methodscomprise administering to the subject a composition comprising bacteriaof the Akkermansia (Akk) and Parabacteroides (Pb) genera, or multiplecompositions that together comprise bacteria of the Akkermansia (Akk)and Parabacteroides (Pb) genera. In some embodiments, the compositionscomprise bacteria of the Akkermansia (Akk) and Parabacteroides (Pb)genera. In some embodiments, the bacteria of the Akkermansia (Akk) genuscomprise Akkermansia muciniphila. In some embodiments, the bacteria ofthe Parabacteroides (Pb) genus comprise Parabacteroides merdae and/orParabacteroides distasonis. In some aspects, the methods comprisedepleting the gut microbiota of the subject and administering acomposition comprising bacteria of Akkermansia genus (e.g., Akkermansiamuciniphila) and Parabacteroides genus (e.g., Parabacteroides merdae orParabacteroides distasonis) to the subject. In some embodiments, atleast 10%, at least 30%, at least 50%, at least 70%, or at least 90% ofthe bacteria in the composition are Akkermansia (Akk) bacteria. In someembodiments, at least 10%, at least 30%, at least 50%, at least 70%, orat least 90% of the bacteria in the composition are Parabacteroides (Pb)bacteria. In some embodiments, the subject is on a diet, and the dietmay be a control diet, a ketogenic diet, a high fat diet, or a lowcarbohydrate diet. The composition may be formulated for oral or rectaldelivery. The composition may be a food product. In some embodiments,the food product is a dairy product (e.g., yogurt). In some embodiments,the composition comprises probiotics. In some embodiments, thecomposition is self-administered. In some embodiments, the compositioncomprises a fecal sample (e.g., a fecal sample from a fecal bank)comprising bacteria of the Akkermansia genus (e.g., Akkermansiamuciniphila) and bacteria of the Parabacteroides (Pb) genus (e.g.,Parabacteroides merdae or Parabacteroides distasonis). In someembodiments, the subject is given antibiotics to deplete the subject'sgut microbiota.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 has six parts, A-F, which show seizure protection and ketogenesisin response to the ketogenic diet correlates with alterations in the gutmicrobiota. Part A shows seizure thresholds in response to 6-Hzstimulation in independent cohorts of mice fed the control diet (CD) orketogenic diet (KD) for 2, 4, 8, 10 or 14 days (left). n=8, 6, 9, 20, 6(CD); 8, 7, 12, 21, 5 (KD). Behavior in representative cohort ofseizure-tested mice at 14 days post dietary treatment (right). Dashedline at y=10 seconds represents threshold for scoring seizures, andtriangle at 24 mA denotes starting current per experimental cohort.n=16. Part B shows levels of serum glucose in independent cohorts ofmice fed CD or KD for 2, 4, 8, 10 or 14 days. Data are normalized toserum glucose levels seen in SPF CD mice for each time point. n=8, 5, 8,8, 19 (CD); 8, 8, 8, 7, 19 (KD). Part C shows levels of serumbeta-hydroxybutyrate (BHB) in independent cohorts of mice fed CD or KDfor 2, 4, 8, 10 or 14 days. n=8, 13, 8, 8, 37 (CD); 8, 16, 8, 7, 38(KD). Part D shows Principal coordinates analysis (PCoA) of weighted(left) and unweighted (right) UniFrac distance matrices based on 16SrDNA profiling of feces from independent cohorts of mice fed CD or KDfor 0, 4, 8 or 14 days. n=3 cages (9 mice)/group. Part E shows Alphadiversity of fecal 16S rDNA sequencing data from mice fed CD or KD for14 days. n=3 cages/group. Part F shows the relative abundance ofAkkermansia muciniphila and Parabacteroides spp. from fecal 16S rDNAsequencing data. n=3 cages (9 mice)/group. Data are presented asmean±s.e.m. Two-way ANOVA with Bonferroni (a-c, e), Kruskal-Wallis withBonferroni (f): P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n.s.=notstatistically significant. SPF=specific pathogen-free(conventionally-colonized), CD=control diet, KD=ketogenic diet,CC50=current intensity producing seizures in 50% of mice tested,BHB=beta-hydroxybutyrate, OTUs=operational taxonomic units.

FIG. 2 has two parts, A-B, which show the ketogenic diet enriches selectbacterial species that differentiate the KD vs. CD gut microbiota. PartA shows the Alpha diversity based on 16S rDNA sequencing of the fecalgut microbiota on days 0, 4, 8, and 14 after treatment with the CD (top)vs. KD (bottom) n=3 per time point. Part B shows the relative abundancesof select bacterial taxa that are enriched in SPF mice fed KD (top row)or CD (bottom row). n=3. Data are presented as mean+s.e.m.Kruskal-Wallis with Bonferroni: *P<0.05, **P<0.01, ***P<0.0001. n.s.=not statistically significant. CD=control diet, KD=ketogenic diet.

FIG. 3 has four parts, A-D, which show the relationship of gutmicrobiota to the anti-seizure effects of the ketogenic diet. Part Ashows seizure thresholds in response to 6-Hz stimulation in SPF, GF orconventionalized GF mice fed CD or KD. n=13, 18, 12, 6. Part B showsserum BHB (left) and glucose (right) levels in SPF, GF orconventionalized GF mice fed CD or KD. n=37, 38, 19, 8. Part C showsseizure thresholds in response to 6-Hz stimulation in SPF mice treatedwith vehicle or Abx pre-dietary treatment. n=13, 18, 13. Part D showsserum BHB (left) and glucose (right) levels in SPF mice treated withvehicle or Abx pre-dietary treatment. n=18, 18, 19 (BHB); n=12, 11, 11(glucose). Data are presented as mean±s.e.m. One-way ANOVA withBonferroni: *P<0.05, **P<0.01, ***P<0.001,****P<0.0001. n.s.=notstatistically significant. SPF=specific pathogen-free(conventionally-colonized), GF=germ-free, GF-conv=germ-freeconventionalized with SPF microbiota, CD=control diet, KD=ketogenicdiet, CC50=current intensity producing seizures in 50% of mice tested.BHB=betahydroxybutyrate, veh=vehicle, Abx=antibiotics (ampicillin,vancomycin, neomycin, metronidazole [AVNM]).

FIG. 4 has two parts, A-B, which show that the KD-associated bacteriasufficiently mediated the anti-seizure effects of the ketogenic diet.Part A shows seizure thresholds in response to 6-Hz stimulation in SPFmice pre-treated with vehicle or Abx, and colonized with Parabacteroidesspp. (P. merdae and P. distasonis), Akkermansia muciniphila, both, orBifidobacterium longum (left). n=13, 18, 15, 6, 8, 5, 5. Behavior inrepresentative cohort of seizure-tested mice (right). Dashed line aty=10 seconds represents the threshold for scoring seizures, and triangleat 24 mA denotes the starting current per experimental cohort. n=12, 16,8, 25. Part B shows seizure thresholds in response to 6-Hz stimulationin GF mice colonized with Parabacteroides spp. (P. merdae and P.distasonis) 0and/or Akkermansia muciniphila (top). n=15, 4, 9, 9.Behavior in seizure-tested mice (bottom). Dashed line at y=10 secondsrepresents threshold for scoring seizures, and triangle at 24 mA denotesstarting current per experimental cohort. n=17, 19. Data are presentedas mean±s.e.m. One-way ANOVA with Bonferroni: **P<0.01, ***P<0.001,****P<0.0001. SPF=specific pathogen-free (conventionally-colonized),GF=germ-free, CD=control diet, KD=ketogenic diet, CC50=current intensityproducing seizures in 50% of mice tested, veh=vehicle, Abx=pre-treatedwith antibiotics (ampicillin, vancomycin, neomycin, metronidazole[AVNM]), Pb=Parabacteroides spp. (P. merdae and P. distasonis),Akk=Akkermansia muciniphila, AkkPb=A. muciniphila, P. merdae and P.distasonis, Bf=Bifidobacterium longum.

FIG. 5 has three parts, A-C, which show KD-associated microbiota conferseizure protection in mice fed the control diet. Part A shows theseizure thresholds in response to 6-Hz stimulation in Abx-treated SPFtransplanted with the CD microbiota (CD-FMT) or KD microbiota (KD-FMT)and fed the CD or KD (left). n=6, 5, 5. Behavior in representativecohort of seizure-tested mice (right). Dashed line at y=10 secondsrepresents threshold for scoring seizures, and triangle at 24 mA denotesstarting current per experimental cohort. n=12. Part B shows seizurethresholds in response to 6-Hz stimulation in SPF mice pre-treated withvehicle or Abx, and colonized with Parabacteroides spp. (P. merdae andP. distasonis), Akkermansia muciniphila, both, or Bifidobacterium longum(left). n=13, 18, 9, 8, 6, 6. Part C shows the seizure thresholds inresponse to 6-Hz stimulation in SPF mice orally gavaged with Akkermansiamuciniphila, P. merdae and P. distasonis (AkkPb), A. muciniphila alone(Akk), or heat-killed Akkermansia muciniphila and Parabacteroides spp(hk-AkkPb) (left). n=6, 6, 4, 3. Data are presented as mean±s.e.m.One-way ANOVA with Bonferroni: *P<0.05, ***P<0.001, ****P<0.0001.SPF=specific pathogen-free (conventionally-colonized), CD=control diet,KD=ketogenic diet, CC50=current intensity producing seizures in 50% ofmice tested, CD-FMT=transplanted with CD microbiota, KDFMT=transplantedwith KD microbiota, veh=vehicle, Abx=pre-treated with antibiotics(ampicillin, vancomycin, neomycin, metronidazole [AVNM]),Pb=Parabacteroides spp. (P. merdae and P. distasonis), Akk=Akkermansiamuciniphila, AkkPb=A. muciniphila, P. merdae and P. distasonis,Bf=Bifidobacterium longum, hk-AkkPb=heat-killed A. muciniphila, P.merdae and P. distasonis.

FIG. 6 has four parts, A-D, which show the reversion of the KDmicrobiota and KD-associated seizure protection in response to thecontrol diet. Part A shows Principal coordinates analysis (PCoA) ofweighted UniFrac distance matrices based on longitudinal 16S rDNAprofiling of feces from SPF mice fed CD for 28 days (CD), mice fed KDfor 28 days (KD), or mice fed KD for 14 days followed by CD for 14 days(KD-CD). n=3 cages/group. Part B shows seizure thresholds in response to6-Hz stimulation in SPF mice fed CD for 28 days (CD), mice fed KD for 28days (KD), or mice fed KD for 14 days followed by CD for 14 days (KD-CD)(left). n=4. Part C shows seizure thresholds in response to 6-Hzstimulation in SPF mice at 21 days after probiotic treatment withAkkermansia muciniphila, P.merdae and P. distasonis (AkkPb), A.muciniphila alone (Akk), or heat-killed Akkermansia muciniphila andParabacteroides spp (hk-AkkPb). (left). n=8. Part D shows seizurethresholds in response to 6-Hz stimulation in SPF mice orally gavagedfor 4 days with vehicle or Akkermansia muciniphila, P. merdae and P.distasonis (AkkPb). n=6, 7, 7, 7. Data are presented as mean±s.e.m.One-way ANOVA with Bonferroni: *P<0.05, **P<0.01, *** *P<0.0001,n.s.=not statistically significant. SPF=specific pathogen-free(conventionally colonized), CD=control diet, KD=ketogenic diet,KD-CD=fed KD for 14 days followed by CD for 14 days. CC50=currentintensity producing seizures in 50% of mice tested, veh=vehicle, AkkAkkermansia muciniphila, AkkPb=A. muciniphila, P. merdae and P.distasonis, hk-AkkPb=heat-killed A. muciniphila, P. merdae and P.distasonis.

FIG. 7 has five parts, A-E, which show KD-associated bacteria mediateprotection against Tonic-Clonic seizures in response to a ketogenicdiet. Part A shows the Principal coordinates analysis (PCoA) of weightedUniFrac distances based on 16S rDNA profiling of feces Kcnal−/− mice fedCD or KD for 14 days. n=5 cages/group. Part B shows the relativeabundances of Akkermansia muciniphila and Parabacteroides spp. fromfecal 16S rDNA sequencing data (right). n=5 cages/group. Part C showsrepresentative EEG trace showing stages used to define seizuresquantified in Part D. Part D shows the average number of seizures perday (left) and total duration of seizures per day (right) in SPFKcnal−/− mice treated with vehicle or Abx, colonized with A. muciniphilaand Parabacteroides spp. or nothing, and fed CD or KD. n=2, 8, 6, 12, 9,3. Part E shows the average number of seizures per day (left), averageduration per seizure (middle) and total duration of seizures per day(right) in SPF CD Kcnal^(−/−) mice treated with GGsTop. Data for SPF CDmice are as in (D). n=6, 4. Data are presented as mean±s.e.m.Kruskal-Wallis with Bonferroni (A, B), non-parametric one-way nestedANOVA with Dunn (D), non-parametric Kolgomorov-Smirnov t test (E).SPF=specific pathogen-free (conventionally-colonized), CD=control diet,KD=ketogenic diet, veh=vehicle, Abx=pre-treated with antibiotics(ampicillin, vancomycin, neomycin, metronidazole [AVNM]), AkkPb=A.muciniphila, P. merdae and P. distasonis.

FIG. 8 has four parts, A-D, which show the association of the reductionsin peripheral gamma-glutamyl amino acids and increases in hippocampalGABA/Glutamate ratios with diet and microbiota-dependent seizureprotection. Part A shows principal components analysis of coloniclumenal metabolites (top) and serum metabolites (bottom) from SPF micefed CD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonizedmice fed KD. n=8 cages/group. Part B shows the levels ofgamma-glutamylated amino acids and cysteine in colonic lumenal contentsfrom SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD, andAkkPb-colonized mice fed KD. n=8 cages/group. Part C shows the levels ofgamma-glutamyl amino acids and glutamine in sera from SPF mice fed CD,SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized mice fedKD. n=8 cages/group. Part D shows the Levels of GABA/glutamate (left)and glutamine (right) in hippocampi of SPF mice fed CD, SPF mice fed KD,Abx-treated mice fed KD, and AkkPb-colonized mice fed KD. n=5. Data arepresented as mean±s.e.m. Two-way ANOVA contrasts (A-C), One-way ANOVAwith Bonferroni (D): *P<0.05, **P<0.01, ***P<0.001,****P<0.0001.n.s.=not statistically significant. CD=control diet, KD=ketogenic diet,SPF=specific pathogen-free (conventionally-colonized), veh=vehicle,Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin,metronidazole [AVNM]), AkkPb=A. muciniphila, P. merdae and P.distasonis, a.u.=arbitrary units.

FIG. 9 has four parts, A-D, which show the modulation of the coloniclumenal and serum metabolomes by the ketogenic diet and microbiotastatus. Part A shows the number of statistically significant alterationsin metabolites out of the 622 detected in colonic lumen and 670 detectedin serum. Values noted in black text are total number of m0etabolitesaltered, with values in green denoting upregulation and values in reddenoting downregulation. n=8 cages/group. Part B shows the levels ofglucose (left) and BHB (right) detected by metabolomics screening ofcolonic lumen (top) and serum (bottom). n=8 cages/group. Part C showsthe levels of non-gamma glutamylated amino acids in colonic lumenalcontents from SPF mice fed CD, SPF mice fed KD, Abx-treated mice fed KD,and AkkPb-colonized mice fed KD. n=8 cages/group. Part D shows thelevels of non-gamma glutamylated amino acids in sera from SPF mice fedCD, SPF mice fed KD, Abx-treated mice fed KD, and AkkPb-colonized micefed KD. n=8 cages/group. Data are presented as mean±s.e.m. Two-way ANOVAcontrasts: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n.s.=notstatistically significant. CD=control diet, KD=ketogenic diet,SPF=specific pathogen-free (conventionally-colonized), veh=vehicle,Abx=pre-treated with antibiotics (ampicillin, vancomycin, neomycin,metronidazole [AVNM]), AkkPb=A. muciniphila, P. merdae and P.distasonis, a.u.=arbitrary units, BHB=beta-hydroxybutyrate.

FIG. 10 has eight parts, A-H, which show that the ketogenic diet andbacterial cross-feeding reduces gamma-glutamyltranspeptidase (GGT)activity, which sufficiently confers seizure protection. Part A showsthe 6-Hz seizure thresholds in response to oral gavage with the GGTinhibitor, GGsTop, in SPF mice fed CD (left). n=6, 9. Behavior inseizure-tested mice (right). Dashed line at y=10 seconds representsthreshold for scoring seizures, and triangle at 24 mA denotes startingcurrent per experimental cohort. n=16. Part B shows the 6-Hz seizurethresholds in response to supplementation with ketogenic amino acids inAbx-treated SPF mice enriched for A. muciniphila and Parabacteroides spp(left). n=5, 6. Behavior in seizure-tested mice (right). Dashed line aty=10 seconds represents threshold for scoring seizures, and triangle at24 mA denotes starting current per experimental cohort. n=12. Part Cshows the total GGT activity per 100 mg feces from SPF CD, SPF KD, AkkPbKD, or AkkPb CD mice (left), and inhibition by GGsTop (right). n=5. PartD shows the total GGT activity per 100 mg feces from SPF CD animalstreated with vehicle, A. muciniphila and Parabacteroides spp. probiotic,or heat-killed bacteria for bi-daily for 28 days (left), and inhibitionby GGsTop (right). n=5. Part E shows the levels of live A. muciniphila(Akk) after incubation in CD vs KD culture media or in CD or KD agaroverlaid with M9 minimal media containing live Parabacteroides merdae(PbM) or no bacteria (0). n=3. Part F shows the levels of live PbM afterincubation in M9 minimal media overlaid on CD or KD agar containing Akkor no bacteria (0). n=5. Part G shows GGT activity in P. merdae grown inM9 media overlaid on CD agar containing A. muciniphila or no bacteria att=24 hrs, and inhibition of GGT activity by GGsTop. n=5. Part H showsGGT activity in P. merdae grown in M9 media overlaid on KD agarcontaining A. muciniphila or no bacteria at t=24 hrs, and inhibition ofGGT activity by GGsTop. n=5. Data are presented as mean±s.e.m.Students't-test (a,b), Two-way ANOVA with Bonferroni (C, D), One-wayANOVA with Bonferroni (E-H): **P<0.01, ***P<0.001, ****P<0.0001.SPF=specific pathogen-free (conventionally-colonized), CD=control diet,KD=ketogenic diet, CC50=current intensity producing seizures in 50% ofmice tested, AA=amino acids, veh=vehicle, Abx=pre-treated withantibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]),AkkPb=A. muciniphila, P. merdae and P. distasonis, GGsTop=GGT inhibitor,PbM=Parabacteroides merdae, Akk=Akkermansia muciniphila, M9=minimalmedia, GGT=gamma-glutamyltranspeptidase, AU=absorbance units.

FIG. 11 has two parts, A and B, which show the amino acid effects onseizures, bacterial GGT activity and dietary modulation of bacterialgenes for amino acid metabolism. Part A shows GGT activity inconventionally-cultured Parabacteroides merdae (left) and A. muciniphila(right), treated with GGsTop or vehicle. n=5. Part B shows the levels ofA. muciniphila (Akk) after 0, 7,or 24 hours incubation in CD agaroverlaid with P. merdae (PbM) that was pre-treated with vehicle orGGsTop in M9 minimal media. n=3. Data are presented as mean±s.e.m.One-way ANOVA with Bonferroni (a): *P<0.05, Two-way ANOVA withBonferroni (b): **P<0.01, PbM=Parabacteroides merdae, Akk Akkermansiamuciniphila, GGsTop=GGT inhibitor, GGT=gamma-glutamyltranspeptidase,AU=absorbance units.

DETAILED DESCRIPTION

Provided herein are methods and compositions for mimicking the effectsof a ketogenic diet by administering probiotic compositions. In certainembodiments, the methods and compositions are for the treatment orprevention of seizures in a subject (e.g., a subject with aneurodevelopmental disorder, such as an autism spectrum disorder, Rettsyndrome, fragile X, attention deficit disorder (ADD),attention-deficit/hyperactivity disorder (ADHD), refractory epilepsy,and/or non-refractory epilepsy). In other embodiments, the methods andcompositions are for preventing or treating a condition (e.g., autismspectrum disorder, epilepsy, seizures, Alzheimer's disease, Huntington'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),cancer, stroke, a metabolic disease (e.g., obesity or diabetes), amitochondrial disorder, depression, migraines (e.g., chronic migraines),Rett syndrome, attention deficit disorder, fragile X syndrome, ortraumatic brain injury (TBI)) in a subject. In preferred embodiments,the methods comprise administering to the subject a compositioncomprising bacteria of Akkermansia genus (e.g., Akkermansia muciniphila)and Parabacteroides genus (e.g., Parabacteroides merdae orParabacteroides distasonis), or multiple compositions that togethercomprise bacteria of Akkermansia genus (e.g., Akkermansia muciniphila)and Parabacteroides genus (e.g., Parabacteroides merdae orParabacteroides distasonis). In other embodiments, the methods andcompositions alter neurotransmitter biosynthesis in a subject. Incertain embodiments, the methods and compositions alter serum ketogenicamino acids in a subject. In other embodiments, the methods andcompositions decrease gamma-glutamyltranspeptidase activity in asubject. In certain embodiments, the methods and compositions decreaseglutamine synthase activity in a subject. In other embodiments, themethods and compositions decrease gamma-glutamyl amino acids in asubject. In certain embodiments, the methods and compositions increaseGABA/glutamate ratio levels in a subject. In other embodiments, themethods and compositions increase glutamine levels in a subject. Inother embodiments, the methods comprise depleting the gut microbiota ofthe subject and administering a composition comprising bacteria ofAkkermansia and Parabacteroides genera to the subject, or multiplecompositions that together comprise bacteria of Akkermansia andParabacteroides genera.

Definitions

As used herein in the specification, “a” or “an” may mean one or more.As used herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence, is well understood in the art,and includes administration of a composition which reduces the frequencyof, or delays the onset of, symptoms of a medical condition in a subjectrelative to a subject which does not receive the composition. Thus,prevention of seizures includes, for example, reducing the number ofseizures in a population of patients receiving a prophylactic treatmentrelative to an untreated control population, and/or delaying theappearance of detectable lesions in a treated population versus anuntreated control population, e.g., by a statistically and/or clinicallysignificant amount.

The term “prophylactic” or “therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “subject” refers to a mammal, including, but not limited to, ahuman or non-human mammal, such as a bovine, equine, canine, ovine, orfeline.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in a manner to improve or stabilize a subject'scondition.

Therapeutic Methods

The disclosure herein, relates, in part, to the discovery that theketogenic diet (KD) induces substantial changes in the gut microbiome,and that enriching KD-associated bacteria via probiotic administration,fecal transplant, or selective microbial reconstitution of the nativemicrobiome mimics the beneficial effects of the KD. Provided herein aremethods and compositions that can replace the KD diet in the treatmentor prevention of a condition as described. The methods and compositionsdescribed herein can be used separately or in conjunction with the KDdiet in the treatment or prevention of a condition described herein.

In certain embodiments, the methods treat or prevent seizures in asubject. In some embodiments, the methods comprise administering acomposition comprising bacteria of Akkermansia and Parabacteroidesgenera. In other embodiments, the methods alter neurotransmitterbiosynthesis in a subject. In certain embodiments, the methods alterserum ketogenic amino acids in a subject. In other embodiments, themethods decrease gamma-glutamyltranspeptidase activity in a subject. Incertain embodiments, the methods decrease glutamine synthase activity ina subject. In other embodiments, the methods decrease gamma-glutamylamino acids in a subject. In certain embodiments, the methods increaseGABA/glutamate ratio levels in a subject. In other embodiments, themethods increase glutamine levels in a subject. In other embodiments,the methods provided herein comprise depleting the gut microbiota of thesubject and administering a composition comprising bacteria ofAkkermansia genus (e.g., Akkermansia muciniphila) and Parabacteroidesgenus (e.g., Parabacteroides merdae or Parabacteroides distasonis) tothe subject. In some embodiments, the subject has epilepsy (e.g.,refractory or non-refractory epilepsy). In some embodiments, the subjecthas a neurodevelopmental disorder. Representative neurodevelopmentaldisorders include autism spectrum disorder, Rett syndrome, fragile X,attention deficit disorder, and attention-deficit/hyperactivitydisorder. In some embodiments, the neurodevelopmental disorder is adisorder known to be comorbid with seizures.

In other embodiments, the subject has a condition responsive to aketogenic diet. The condition may be Alzheimer's disease, Huntington'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),cancer, stroke, a metabolic disease, a mitochondrial disorder,depression, migraines (e.g., chronic migraines), or traumatic braininjury (TBI). In some embodiments, the methods and compositions compriseadministering to the subject a composition provided herein. In someembodiments, the condition is epilepsy, seizures, autism spectrumdisorder, Alzheimer's disease, Huntington's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), cancer, stroke, ametabolic disease (e.g., obesity or diabetes) , a mitochondrialdisorder, depression, migraines (e.g., chronic migraines), Rettsyndrome, attention deficit disorder, fragile X syndrome, or traumaticbrain injury (TBI). In some embodiments, the compositions and methodsprovided herein are useful in treating or preventing aging oraging-associating conditions. In some embodiments, the compositions andmethods provided herein can replace the ketogenic diet in the treatmentor prevention of a condition described herein; in other embodiments, thecompositions and methods provided herein can be combined with theketogenic diet. More information on conditions may be found in Stafstromet al. (2012) Front. Pharmacol. 3:59, hereby incorporated in itsentirety.

The composition may be formulated for oral delivery. In someembodiments, the composition may comprise probiotics. In someembodiments, the compositions disclosed herein are food products. Thecomposition may be in the form of a pill, tablet, or capsule. In someembodiments, the subject may be a mammal (e.g., a human). In someembodiments, the composition is self-administered. While it is preferredfor a single composition to comprise all the bacteria to beadministered, it will be recognized that for any of the variousembodiments described herein, the combination of bacteria can similarlybe administered in multiple compositions that together comprise thecombination of bacteria. For example, the invention further provideskits comprising multiple compositions that together comprises bacteriaof Akkermansia genus (e.g., Akkermansia muciniphila) and Parabacteroidesgenus (e.g., Parabacteroides merdae or Parabacteroides distasonis).

In some embodiments, the composition is formulated for rectal delivery(e.g., a fecal sample). In some embodiments, the subject undergoes fecalmicrobiota transplant, wherein the transplant comprises a compositiondisclosed herein. Fecal microbiota transplantation (FMT), also commonlyknown as ‘fecal bacteriotherapy’ represents a therapeutic protocol thatallows the reconstitution of colon microbial communities. The processinvolves the transplantation of fecal bacteria from a healthy individualinto a recipient. FMT restores colonic microflora by introducing healthybacterial flora through infusion of a fecal sample, e.g., by enema,orogastric tube or by mouth in the form of a capsule containingfreeze-dried material, obtained from a healthy donor. In someembodiments, the fecal sample is from a fecal bank.

In some embodiments, the bacterial DNA in subject's gut microbiota issequenced. The subject's gut bacterial DNA may be sequenced prior toadministration of the composition. For example, a sample comprisingbacterial DNA may be obtained from the subject, and the bacterial DNA isthen sequenced for Akkermansia (Akk) and/or Parabacteroides DNA,therefore measuring the presence or level of Akkermansia and/orParabacteroides in the subject's gut microbiota. The compositiondisclosed herein may then be administered to the subject if the level ofAkkermansia and/or Parabacteroides is low. In some embodiments, thesubject is deemed to have low levels of Akkermansia and/orParabacteroides if less than 0.0001%, less than 0.001%, less than 0.01%,less than 0.02%, less than 0.03%, less than 0.04%, less than 0.05%, lessthan 0.06% less than 0.07%, less than 0.08%, less than 0.09%, less than0.1%, less than 0.2%, less than 0.3% less than 0.4%, less than 0.5%,less than 0.6%, less than 0.7%, less than .8%, less than 0.9%, less than1%, less than 2%, less than 3%, less than 5%, less than 7%, less than10%, less than 20%, less than 30%, less than 40%, or less than 50% ofthe bacteria in the sample is Akkermansia and/or Parabacteroides DNA.Bacterial DNA to be sequenced may be obtained through any means known inthe art, including, but not limited to, obtaining a fecal sample fromthe subject and isolating the bacterial DNA. Bacterial DNA sequencing byany known technique in the art, including, but not limited to, MaxamGilbert sequencing, Sanger sequencing, shotgun sequencing, bridge PCR,or next generation sequencing methods, such as massively parallelsignature sequencing (MPSS), polony sequencing, 454 pyrosequencing,Illumina (Solexa) sequencing, SOLiD sequencing, Ion torrentsemiconductor sequencing, DNA nanoball sequencing, heliscope singlemolecule sequencing, single molecule real time (SMRT) sequencing, ornanopore DNA sequencing.

In some embodiments, the above methods directly act to reduce the amountof pathogenic bacteria in a subject (i.e., in the gastrointestinal tractof the subject). In some embodiments, this includes any such therapythat achieves the same goal of reducing the number of pathogenicorganisms, when used in combination with the compositions describedherein, would lead to replacement of the pathogenic microflora involvedin the diseased state with natural microflora associated with anon-diseased state, or less pathogenic species occupying the sameecological niche as the type causing a disease state. For example, asubject may undergo treatment with antibiotics (e.g., antimicrobialcompounds) or a composition comprising antibiotics to target anddecrease the prevalence of pathogenic organisms, and subsequently betreated with a composition described herein. The treatment may alsocomprise an antifungal or anti-viral compound.

Suitable antimicrobial compounds include capreomycins, includingcapreomycin IA, capreomycin IB, capreomycin IIA and capreomycin IIB;carbomycins, including carbomycin A; carumonam; cefaclor, cefadroxil,cefamandole, cefatrizine, cefazedone, cefazolin, cefbuperazone,cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefime, ceftamet,cefmenoxime, cefmetzole, cefminox, cefodizime, cefonicid, cefoperazone,ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpimizole,cefpiramide, cefpirome, cefprozil, cefroxadine, cefsulodin, ceftazidime,cefteram, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone,cefuroxime, cefuzonam, cephalexin, cephalogycin, cephaloridine,cephalosporin C, cephalothin, cephapirin, cephamycins, such ascephamycin C, cephradine, chlortetracycline; chlarithromycin,clindamycin, clometocillin, clomocycline, cloxacillin, cyclacillin,danofloxacin, demeclocyclin, destomycin A, dicloxacillin, dirithromycin,doxycyclin, epicillin, erythromycin A, ethanbutol, fenbenicillin,flomoxef, florfenicol, floxacillin, flumequine, fortimicin A, fortimicinB, forfomycin, foraltadone, fusidic acid, gentamycin, glyconiazide,guamecycline, hetacillin, idarubicin, imipenem, isepamicin, josamycin,kanamycin, leumycins such as leumycin A1, lincomycin, lomefloxacin,loracarbef, lymecycline, meropenam, metampicillin, methacycline,methicillin, mezlocillin, micronomicin, midecamycins such as midecamycinA1, mikamycin, minocycline, mitomycins such as mitomycin C, moxalactam,mupirocin, nafcillin, netilicin, norcardians such as norcardian A,oleandomycin, oxytetracycline, panipenam, pazufloxacin, penamecillin,penicillins such as penicillin G, penicillin N and penicillin O,penillic acid, pentylpenicillin, peplomycin, phenethicillin, pipacyclin,piperacilin, pirlimycin, pivampicillin, pivcefalexin, porfiromycin,propiallin, quinacillin, ribostamycin, rifabutin, rifamide, rifampin,rifamycin SV, rifapentine, rifaximin, ritipenem, rekitamycin,rolitetracycline, rosaramicin, roxithromycin, sancycline, sisomicin,sparfloxacin, spectinomycin, streptozocin, sulbenicillin, sultamicillin,talampicillin, teicoplanin, temocillin, tetracyclin, thostrepton,tiamulin, ticarcillin, tigemonam, tilmicosin, tobramycin,tropospectromycin, trovafloxacin, tylosin, and vancomycin, and analogs,derivatives, pharmaceutically acceptable salts, esters, prodrugs, andprotected forms thereof.

Suitable anti-fungal compounds include ketoconazole, miconazole,fluconazole, clotrimazole, undecylenic acid, sertaconazole, terbinafine,butenafine, clioquinol, haloprogin, nystatin, naftifine, tolnaftate,ciclopirox, amphotericin B, or tea tree oil and analogs, derivatives,pharmaceutically acceptable salts, esters, prodrugs, and protected formsthereof.

Suitable antiviral agents include acyclovir, azidouridine, anismoycin,amantadine, bromovinyldeoxusidine, chlorovinyldeoxusidine, cytarabine,delavirdine, didanosine, deoxynojirimycin, dideoxycytidine,dideoxyinosine, dideoxynucleoside, desciclovir, deoxyacyclovir,efavirenz, enviroxime, fiacitabine, foscamet, fialuridine,fluorothymidine, floxuridine, ganciclovir, hypericin, idoxuridine,interferon, interleukin, isethionate, nevirapine, pentamidine,ribavirin, rimantadine, stavudine, sargramostin, suramin, trichosanthin,tribromothymidine, trichlorothymidine, trifluorothymidine, trisodiumphosphomonoformate, vidarabine, zidoviridine, zalcitabine and3-azido-3-deoxythymidine and analogs, derivatives, pharmaceuticallyacceptable salts, esters, prodrugs, and protected forms thereof.

Other suitable antiviral agents include 2′,3′-dideoxyadenosine (ddA),2′,3′-dideoxyguanosine (ddG), 2′,3′-dideoxycytidine (ddC),2′,3′-dideoxythymidine (ddT), 2′3′-dideoxy-dideoxythymidine (d4T),2′-deoxy-3′-thia-cytosine (3TC or lamivudime),2′,3′-dideoxy-2′-fluoroadenosine, 2′,3′-dideoxy-2′-fluoroinosine,2′,3′-dideoxy-2′-fluorothymidine, 2′,3′-dideoxy-2′-fluorocytosine,2′3′-dideoxy-2′,3′-didehydro-2′-fluorothymidine (Fd4T),2′3′-dideoxy-2′-beta-fluoroadenosine (F-ddA),2′3′-dideoxy-2′-beta-fluoro-inosine (F-ddl), and2′,3′-dideoxy-2′-beta-flurocytosine (F-ddC). In some embodiments, theantiviral agent is selected from trisodium phosphomonoformate,ganciclovir, trifluorothymidine, acyclovir, 3′-azido-3′-thymidine (AZT),dideoxyinosine (ddl), and idoxuridine and analogs, derivatives,pharmaceutically acceptable salts, esters, prodrugs, and protected formsthereof.

Compositions

In some aspects, the invention relates to a composition (e.g., a foodproduct or a pharmaceutical composition) comprising bacteria ofAkkermansia (Akk) and Parabacteroides (Pb) genera. The composition maycomprise a pharmaceutically acceptable carrier. The composition maycomprise probiotics. The pharmaceutical compositions disclosed hereinmay be delivered by any suitable route of administration, includingorally, bucally, sublingually, parenterally, and rectally, as bypowders, ointments, drops, liquids, gels, tablets, capsules, pills, orcreams. In certain embodiments, the pharmaceutical compositions aredelivered generally (e.g., via oral administration). In certain otherembodiments, the compositions disclosed herein are delivered rectally.

In certain embodiments, the invention provides kits comprising multiplecompositions that together comprises bacteria of Akkermansia genus(e.g., Akkermansia muciniphila) and Parabacteroides genus (e.g.,Parabacteroides merdae or Parabacteroides distasonis) (e.g., that, ifcombined, would result in a composition as described anywhere in thissection).

The composition may comprise any species of Parabacteroides, including,but not limited to, P. chartae, P. chinchillae, P. distasonis, P.faecis, P. goldsteinii, P. gordonii, P. johnsonii, or P. merdae. In someembodiments, at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35% , at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, of the bacteria in the composition are Parabacteroides (Pb)bacteria. The bacteria of Akkermansia in the composition may compriseAkkermansia muciniphila. In some embodiments, the compositions disclosedherein may comprise at least 1%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% Akkermansia bacteria.

Compositions described herein may be used for oral administration to thegastrointestinal tract, directed at the objective of introducing thebacteria (e.g., the bacteria disclosed herein) to tissues of thegastrointestinal tract. The formulation for a composition (e.g., aprobiotic composition) of the present invention may also include otherprobiotic agents or nutrients which promote spore germination and/orbacterial growth. An exemplary material is a bifidogenicoligosaccharide, which promotes the growth of beneficial probioticbacteria. In some embodiments, the probiotic bacterial composition isadministered with a therapeutically-effective dose of an (preferably,broad spectrum) antibiotic, or an anti-fungal agent. In someembodiments, the compositions described herein are encapsulated into anenterically-coated, time-released capsule or tablet. The enteric coatingallows the capsule/tablet to remain intact (i.e., undissolved) as itpasses through the gastrointestinal tract, until after a certain timeand/or until it reaches a certain part of the GI tract (e.g., the smallintestine). The time-released component prevents the “release” of theprobiotic bacterial strain in the compositions described herein for apre-determined time period.

The composition may be a food product, such as, but not limited to, adairy product. The dairy product may be cultured or a non-cultured(e.g., milk) dairy product. Non-limiting examples of cultured dairyproducts include yogurt, cottage cheese, sour cream, kefir, buttermilk,etc. Dairy products also often contain various specialty dairyingredients, e.g. whey, non-fat dry milk, whey protein concentratesolids, etc. The dairy product may be processed in any way known in theart to achieve desirable qualities such as flavor, thickening power,nutrition, specific microorganisms and other properties such as moldgrowth control. The compositions of the present invention may alsoinclude known antioxidants, buffering agents, and other agents such ascoloring agents, flavorings, vitamins, or minerals.

In some embodiments, the compositions of the present invention arecombined with a carrier (e.g., a pharmaceutically acceptable carrier)which is physiologically compatible with the gastrointestinal tissue ofthe subject(s) to which it is administered. Carriers can be comprised ofsolid-based, dry materials for formulation into tablet, capsule orpowdered form; or the carrier can be comprised of liquid or gel-basedmaterials for formulations into liquid or gel forms. The specific typeof carrier, as well as the final formulation depends, in part, upon theselected route(s) of administration. The therapeutic composition of thepresent invention may also include a variety of carriers and/or binders.In some embodiments, the carrier is micro-crystalline cellulose (MCC)added in an amount sufficient to complete the one gram dosage totalweight. Carriers can be solid-based dry materials for formulations intablet, capsule or powdered form, and can be liquid or gel-basedmaterials for formulations in liquid or gel forms, which forms depend,in part, upon the routes of administration. Typical carriers for dryformulations include, but are not limited to: trehalose, malto-dextrin,rice flour, microcrystalline cellulose (MCC) magnesium sterate,inositol, FOS, GOS, dextrose, sucrose, and like carriers. Suitableliquid or gel-based carriers include but are not limited to: water andphysiological salt solutions; urea; alcohols and derivatives (e.g.,methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol,propylene glycol, and the like). Preferably, water-based carrierspossess a neutral pH value (i.e., pH 7.0). Other carriers or agents foradministering the compositions described herein are known in the art,e.g., in U.S. Pat. No. 6,461,607.

In some embodiments, the composition further comprises other bacteria ormicroorganisms known to colonize the gastrointestinal tract. Forexample, the cornposition may comprise species belonging to theFirmicutes phylum, the Proteobacteria phylum, the Tenericutes phylum,the Actinobacteria phylum, or a combination thereof. Examples ofadditional bacteria and microorganisms that may be included in thesubject compositions include, but are not limited to, Saccharomyces,Bacteroides, Eubacterium, Clostridium, Lactobacillus, Fusobacterium,Propionibacterium, Streptococcus, Enteroccus, Lactococcus andStaphylococcus, Peptostreptococcus. In certain embodiments, thecomposition is substantially free of bacteria that increase the risk ofseizures or otherwise detract from the effect of a ketogenic diet. Suchbacteria include Bifidobacterium bacteria. Thus, in some embodiments,the composition is substantially free of Bacteroides bacteria. Acomposition is substantially free of a bacterial type if that type makesup less than 10% of the bacteria in a composition, preferably less than5%, even more preferably less than 1%, most preferably less than 0.5%,or even 0% of the bacteria in the composition.

In some embodiments, the composition comprises a fecal sample comprisingat least one species of Akkermansia (Akk) and at least one species ofParabacteroides (Pb). In some embodiments, the fecal sample is from afecal bank. In some embodiments, the compositions may be added to afecal sample prior to administration to the subject.

In some embodiments, provided herein are methods of treating orpreventing a condition, such as seizures, by administering a composition(e.g., a fecal sample) that is enriched for at least one species ofAkkermansia (Akk) and at least one species of Parabacteroides (Pb) tothe subject. The fecal sample is enriched if at least 0.1%, at least0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.6%, atleast 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, atleast 0.7%, at least 0.8%, at least 0.9%, at least 1%, or at least 2%,at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50% of the bacteria in the fecal sample is Akkermansia (Akk). Insome embodiments, fecal sample is enriched if at least 0.1%, at least0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.6%, atleast 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, atleast 0.7%, at least 0.8%, at least 0.9%, at least 1%, or at least 2%,at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, or atleast 50% of the bacteria in the fecal sample is and Parabacteroides(Pb). In some embodiments, the fecal sample is from a fecal bank. Insome embodiments, the fecal sample is from a donor.

The composition may further comprise a nutrient. In some embodiments,the nutrient aids in the growth of bacteria (e.g., bacteria disclosedherein). In some embodiments, the nutrient is a component listed in FIG.12. In some embodiments, the nutrient is a lipid (e.g., lineoleic acid,stearic acid, or palmitic acid). In some embodiments, the nutrient maybe conjointly administered with a composition disclosed herein. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different agents (e.g., a compositiondisclosed herein and a nutrient disclosed herein) such that the secondagent is administered while the previously administered agent is stilleffective in the body. For example, the compositions disclosed hereinand the nutrients disclosed herein can be administered either in thesame formulation or in a separate formulation, either concomitantly orsequentially.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular agent employed, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldprescribe and/or administer doses of the compounds employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXEMPLIFICATION Example 1 Materials and Methods Animals and Diets

Three to four week old SPF wild type Swiss Webster mice (Taconic Farms),GF wild type Swiss Webster mice (Taconic Farms) and SPF C3HeB/FeJ KCNA1KO mice (Jackson Laboratories) were bred in UCLA's Center for HealthSciences Barrier Facility. Breeding animals were fed “breeder” chow (LabDiets 5K52). Experimental animals were fed standard chow (Lab Diet5010), 6:1 ketogenic diet (Harlan Teklad TD.07797.PWD) or vitamin- andmineral-matched control diet (Harlan Teklad TD.150300). Juvenile micewere used to i) mimic the typical use of the KD to treat pediatric andadolescent epileptic patients, ii) align the timing of mouse braindevelopment to early human brain development, where neurodevelopmentalmilestones in 3-week old mice are comparable to those of the 2-3 yearold human brain⁶⁰, and iii) preclude pre-weaning dietary treatment,where effects of the diet on maternal behavior and physiology wouldconfound direct effects of the diet on offspring. Mice were randomlyassigned to an experimental group. All animal experiments were approvedby the UCLA Animal Care and Use Committee.

6-Hz Psychomotor Seizure Assay

The 6-Hz test was conducted as previously described¹³. Pilot studiesrevealed no sexual dimorphism in seizure threshold. All subsequentexperimental cohorts contained male mice. One drop (˜50 ul) of 0.5%tetracaine hydrochloride ophthalmic solution was applied to the corneasof each mouse 10-15 min before stimulation. Corneal electrodes werecoated with a thin layer of electrode gel (Parker Signagel). Aconstant-current current device (ECT Unit 57800, Ugo Basile) was used todeliver current at 3 sec duration, 0.2 ms pulse-width and 6 pulses/sfrequency. CC50 (the intensity of current required to elicit seizures in50% of the experimental group) was measured as a metric for seizuresusceptibility. Pilot experiments were conducted to identify 24 mA asthe CC50 for SPF wild-type Swiss Webster mice. Each mouse wasseizure-tested only once, and thus at least n>6 mice were used toadequately power each experiment group. To determine CC50s for eachexperimental group, 24 mA currents were administered to the first mouseper experimental group per cohort, followed by fixed increases ordecreases by 2 mA intervals. Mice were restrained manually duringstimulation and then released into a new cage for behavioralobservation. Locomotor behavior was recorded using Ethovision XTsoftware (Noldus) and quantitative measures for falling, taildorsiflexion (Straub tail), forelimb clonus, eye/vibrissae twitching andbehavioral remission were scored manually. For each behavioralparameter, we observed no correlation between percentage incidenceduring 24 mA seizures and microbiota status or group seizuresusceptibility, suggesting a primary effect of the microbiota on seizureincidence rather than presentation or form. Latency to exploration (timeelapsed from when an experimental mouse is released into the observationcage (after corneal stimulation) to its first lateral movement) wasscored using Ethovision and manually with an electronic timer.Within-diet groups were tested blindly. Authentic blinding acrossdifferent-diet groups was not possible due to diet-induced changes instool color. However, results from pilot experiments reveal nosignificant differences between results acquired from the sameexperimental groups tested blinded vs non-blinded. Mice were scored asprotected from seizures if they did not show seizure behavior andresumed normal exploratory behavior within 10 s. Seizure threshold(CC50) was determined as previously described', using the average loginterval of current steps per experimental group, where sample n isdefined as the subset of animals displaying the less frequent seizurebehavior. Data used to calculate CC50 are also displayed as latency toexplore for each current intensity, where n represents the total numberof biological replicates per group regardless of seizure outcome.

Glucose Measurements

Blood samples were collected by cardiac puncture and spun through SSTvacutainers (Becton Dickinson) for serum separation. Glucose levels weredetected in sera by colorimetric assay according to the manufacturer'sinstructions (Cayman Chemical). Data compiled across multipleexperiments are expressed as glucose concentrations normalized to SPFcontrols within each experiment.

Beta-Hydroxybutyrate (BHB) Measurements

Blood was collected by cardiac puncture and spun through SST vacutainers(Becton Dickinson) for serum separation. The colon was washed andflushed with PBS to remove lumenal contents. Frontal cortex,hippocampus, hypothalamus and cerebellum were microdissected and liverswere harvested and washed in PBS. Tissue samples were sonicated on icein 10 s intervals at 20 mV in RIPA lysis buffer (Thermo Scientific). BHBlevels were detected in sera by colorimetric assay according to themanufacturer's instructions (Cayman Chemical). Data were normalized tototal protein content as detected by BCA assay (Thermo Pierce). Datacompiled across multiple experiments are expressed as BHB concentrationsnormalized to SPF controls within each experiment.

16S rDNA Microbiome Profiling

Bacterial genomic DNA was extracted from mouse fecal samples or coloniclumenal contents using the MoBio PowerSoil Kit, where the sample nreflects independent cages containing 3 mice per cage. The library wasgenerated according to methods adapted from ⁶². The V4 regions of the16S rDNA gene were PCR amplified using individually barcoded universalprimers and 30 ng of the extracted genomic DNA. The PCR reaction was setup in triplicate, and the PCR product was purified using the QiaquickPCR purification kit (Qiagen). The purified PCR product was pooled inequal molar concentrations quantified by the Kapa library quantificationkit (Kapa Biosystems, KK4824) and sequenced by Laragen, Inc. using theIllumina MiSeq platform and 2×250 bp reagent kit for paired-endsequencing. Operational taxonomic units (OTUs) were chosen by openreference OTU picking based on 97% sequence similarity to the Greengenes13_5 database. Taxonomy assignment and rarefaction were performed usingQIIME1.8.0⁶³ using 85,134 reads per sample. Metagenomes were inferredfrom closed reference OTU tables using PICRUSt⁶⁴. Results were filteredto display the top 72 genes relevant to amino acid metabolism in FIG.S7C.

Microbiota Conventionalization

Fecal samples were freshly collected from adult SPF Swiss Webster miceand homogenized in pre-reduced PBS at 1 ml per pellet. 100 ul of thesettled suspension was administered by oral gavage to recipient GF mice.For mock treatment, mice were gavaged with pre-reduced PBS.

Antibiotic Treatment

SPF mice were gavaged with a solution of vancomycin (50 mg/kg), neomycin(100 mg/kg) and metronidazole (100 mg/kg) every 12 hours daily for 7days, according to methods previously described by Reikvam et al., PloSone (6), 2011. Ampicillin (1 mg/ml) was provided ad libitum in drinkingwater. For mock treatment, mice were gavaged with normal drinking waterevery 12 hours daily for 7 days. For Kcnal^(−/−) mice, drinking waterwas supplemented with vancomycin (500 mg/ml), neomycin (1 mg/ml) andampicillin (1 mg/ml) for 1 week to preclude the stress of oral gavage inseizure-prone mice.

Gnotobiotic Colonization and Bacterial Enrichment in Antibiotic-TreatedMice

A. muciniphila (ATCC BAA845) was cultured under anaerobic conditions inBrain Heart Infusion (BHI) media supplemented with 0.05% hog gastricmucin type III (Sigma Aldrich). P. merdae (ATCC 43184) and P. distasonis(ATCC 8503) were grown in anaerobic conditions in Reinforced ClostridialMedia (RCM). 10⁹ cfu bacteria were suspended in 200 ul pre-reduced PBSand orally gavaged into antibiotic-treated mice or germ-free mice. Whenco-administered as “A. muciniphila and Parabacteroides spp.”, a ratio of2:1:1 was used for A. muciniphila: P. merdae: P. distasonis. For mocktreatment, mice were gavaged with pre-reduced PBS. Pilot studiesrevealed no significant differences between colonization groups in fecalDNA concentration or 16S rDNA amplification, as measures relevant tobacterial load. Mice were maintained in microisolator cages and handledaseptically. Mice were seizure tested at 14 days after colonization.

Fecal Microbiota Transplant

Fecal samples were freshly collected from donor mice fed KD or CD for 14days and suspended at 50 mg/ml in pre-reduced PBS. Antibiotic-treatedmice were colonized by oral gavage of 100 ul suspension. For mocktreatment, mice were gavaged with pre-reduced PBS. Mice were housed inmicroisolator cages and handled aseptically. Seizure testing wasconducted at 4 days after transplant.

Bacterial Treatment

A. muciniphila, P. merdae and P. distasonis were freshly cultured inanaerobic conditions as described above, and then washed, pelleted andre-suspended at 5×10⁹ cfu/ml in pre-reduced PBS. A. muciniphila withParabacteroides spp. were prepared at a 2:1:1 ratio. For heat-killing,bacteria were placed at 95° C. for 10 min. Mice were gavaged every 12hours for 28 days with 200 ul bacterial suspension or sterilepre-reduced PBS as vehicle control.

Kcnal Seizure Recordings

EEG Implantation and Recovery. EEGs were recorded from male and femaleKcnal^(−/−) mice at 6-7 weeks of age. Kcnal^(+/+) littermates were usedas controls. We observed no significant differences between males andfemales in seizure frequency and duration. Data presented include bothsexes. Mice were anesthetized with isoflurane (5% induction, 2%maintenance) and eye ointment applied to each eye. Fur was removed alongthe head, and the area was cleaned with three alternative scrubs ofchlorohexidine and 70% isopropanol. In a biosafety cabinet, mice werepositioned in a stereotaxic apparatus (Harvard Biosciences) and 1 mg/kglidocaine+1 mg/kg bupivacaine was applied locally along the incisionsite. Using sterile surgical tools, a 2 cm incision was made along thedorsal midline from the posterior margin of the eyes to a point midwaybetween the scapulae. A subcutaneous pocket along the dorsal flank wascreated and the pocket irrigated with sterile saline. A wirelesstelemetry transmitter was inserted with bi-potential leads orientedcranially. The skull was cleaned with 3% hydrogen peroxide followed by70% isopropanol. Using a 1.0 mm micro drill bit, the skull wasperforated to generate two small holes halfway between the bregma andlambda, and 1-2 mm from the sagittal suture. Bilateral EEG recordingelectrodes (Data Sciences International (DSI) PhysioTel, ETA-F10) wereepidurally implanted over the frontoparietal cortex. Sterile acrylic wasapplied to the dried area. The incision site was closed with absorbable5-0 sutures and cleaned with 3% hydrogen peroxide followed by 70%ethanol. Animals were housed individually in autoclaved microisolatorcages and allowed to recover for 3-5 days before recordings wereinitiated.

Data Acquisition and Analysis

During EEG recordings animals were freely moving and maintained onexperimental diet. EEG traces were acquired over 3 days using the DSIPonemah V5.1 data acquisition system. Simultaneous video recordings ofbehavioral seizures were correlated with EEG recordings and scored basedon an adapted Racine scale and defined by 5 stages: 1) myoclonic jerk,2) head stereotypy and facial clonus, 3) bilateral and alternatingforelimb/hindlimb clonus, 4) rearing and falling, and 5) generalizedtonic-clonic episodes. Data were analyzed by a blinded researcher usingNeuroscore CNS Software (DSI). EEG signals were filtered using a 10 Hzhigh pass filter, and seizure events were detected by blinded manualscoring. Seizures were defined as patterns of high-frequency,high-voltage synchronized heterogeneous spike wave forms with amplitudesat least 2-fold greater than background with more than 6 s duration. Thespike frequency was determined as number of spikes occurring abovebaseline in a given seizure, and the interspike interval was analyzed asa function of the time between spikes for five representative seizuresin each phase per mouse. The duration of maximum spike amplitude wasdetermined as the percent time spent in spikes that were three times aslarge as the baseline for five representative seizures in each phase permouse.

Colonic Lumenal and Serum Metabolomics

Samples were collected from mice housed across independent cages, withat least 2 mice per cage. Colonic lumenal contents were collected fromterminal mouse dissections, immediately snap frozen in liquid nitrogenand stored at −80° C. Serum samples were collected by cardiac puncture,separated using SST vacutainer tubes and frozen at -80° C. Samples wereprepared using the automated MicroLab STAR system (Hamilton Company) andanalyzed on GC/MS, LC/MS and LC/MS/MS platforms by Metabolon, Inc.Protein fractions were removed by serial extractions with organicaqueous solvents, concentrated using a TurboVap system (Zymark) andvacuum dried. For LC/MS and LC-MS/MS, samples were reconstituted inacidic or basic LC-compatible solvents containing>11 injection standardsand run on a Waters ACQUITY UPLC and Thermo-Finnigan LTQ massspectrometer, with a linear ion-trap front-end and a Fourier transformion cyclotron resonance mass spectrometer back-end. For GC/MS, sampleswere derivatized under dried nitrogen usingbistrimethyl-silyl-trifluoroacetamide and analyzed on a Thermo-FinniganTrace DSQ fast-scanning single-quadrupole mass spectrometer usingelectron impact ionization. Chemical entities were identified bycomparison to metabolomic library entries of purified standards.Following log transformation and imputation with minimum observed valuesfor each compound, data were analyzed using one-way ANOVA to test forgroup effects. P and q-values were calculated based on two-way ANOVAcontrasts. Principal components analysis was used to visualize variancedistributions. Supervised Random Forest analysis was conducted toidentify metabolomics prediction accuracies.

Hippocampal Metabolomics

Hippocampal tissues were homogenized in 1 ml cold 80% MeOH andvigorously mixed on ice followed by centrifugation (1.3*10⁴ rpm, 4° C.).5 ug supernatant was transferred into a glass vial, supplemented with 5nmol D/L-norvaline, dried down under vacuum, and finally re-suspended in70% acetonitrile. For the mass spectrometry-based analysis of thesample, 51 were injected onto a Luna NH2 (150 mm×2 mm, Phenomenex)column. The samples were analyzed with an UltiMate 3000RSLC (ThermoScientific) coupled to a Q Exactive mass spectrometer (ThermoScientific). The Q Exactive was run with polarity switching (+4.00kV/−4.00 kV) in full scan mode with an m/z range of 70-1050. Separationwas achieved using A) 5 mM NH₄AcO (pH 9.9) and B) ACN. The gradientstarted with 15% A) going to 90% A) over 18 min, followed by anisocratic step for 9 min. and reversal to the initial 15% A) for 7 min.Metabolites were quantified with TraceFinder 3.3 using accurate massmeasurements (≤3 ppm), retention times of pure standards and MS2fragmentation patterns. Data analysis, including principal componentanalysis and hierarchical clustering was performed using R.

Amino Acid Supplementation

Four-week old Swiss Webster SPF mice were treated with antibiotics,colonized with A. muciniphila and Parabacteroides spp., and fed KD for14 days, as described in methods above. Beginning on the evening of day11, mice were injected intraperitoneally every 12 hours for 3 days withketogenic amino acid cocktail (Sigma Aldrich)—L-leucine (2.0 mg/kg),L-lysine (2.0 mg/kg), L-tyrosine (2.4 mg/kg), L-tryptophan (1.6 mg/kg),and L-threonine (3.1 mg/kg) in sterile PBS. Concentrations are based onphysiological levels reported for each amino acid in mouse blood ²⁶ andon fold-changes observed in our metabolomics dataset for each amino acidbetween control SPF CD and AkkPb KD mice (Table S4). Vehicle-treatedmice were injected with PBS (200 ul/30 g mouse). On day 14, mice weretested for 6-Hz seizures 2 hours after the final morning amino acidinjection, with a prior 1-hour acclimation period in the behavioraltesting room.

GGsTop Treatment

For wild type mice: 4-week old SPF Swiss Webster mice were fed CD adlibitum for 14 days. Beginning on the evening of day 11, mice wereorally gavaged every 12 hours with 13.3 mg/kg3-[[(3-amino-3-carboxypropyl)methoxyphosphinyl]oxy]benzeneacetic acid(GGsTop, Tocris Bioscience) in sterile water. Vehicle-treated mice weregavaged with sterile water (200 ul/30 g mouse). On day 14, mice weretested for 6-Hz seizures 2 hours after the final morning GGsTop gavage,with a prior 1-hour acclimation period in the behavioral testing room.For Kcnal mice: 3-4 week old Kcnal^(−/−) mice were fed the CD ad libitumfor 23 days. On Day 15, EEG transmitters were implanted as described inthe Kcnal Seizure Recordings section above. On the evening of day 18,mice were orally gavaged every 12 hours with 13.3 mg/kg GGsTop throughthe morning of day 21. Seizures were recorded over 3 days by EEGbeginning 2 hours after the final gavage.

Cross-Feeding in vitro Assay

Cross-feeding was measured as previously described. A. muciniphila wasembedded at 2×10⁶ cfu/ml in 5 ml pre-reduced CD or KD-based liquid mediasupplemented with 1% agar at the bottom of an anaerobic tube, and P.merdae was overlaid above it at 6×10⁶ cfu/ml in 5 ml pre-reduced M9minimal media. Diet-based media were generated by aseptically suspendingmouse KD vs. CD diets, described above, to 2 kcal/ml in M9 media. Pilotexperiments confirmed no ectopic translocation of embedded A.muciniphila from the agar compartment into the above M9 liquidcompartment. For each time point, aliquots were taken from the top andbottom compartments, plated in a dilution series in rich media (RCM forP. merdae and BHI+0.05% mucins for A. muciniphila), and colonies werecounted. For GGsTop pre-treatment experiments, P. merdae was incubatedwith 500 uM GGsTop vs vehicle in RCM media at 37° C. for 2 hours andthen washed with sterile media. Pilot experiments revealed nosignificant effect of GGsTop pre-treatment on P. merdae viability.

GGT Activity Assay

GGT activity was measured as previously described in van der Stel,Frontiers in Microbiology (6), 567 (2015). For anaerobic cultures,bacteria were seeded at 3×10⁵ cfu/ml in CD- and KD-based media. 1 mlbacterial suspension was pelleted and frozen at −80° C. for 1 hr.Separate aliquots of the same suspension were plated in BHI mucin agarmedia or RCM and incubated at 37° C. in a Coy anaerobic chamber forlater data normalization by bacterial cfu. Pellets were then resuspendedin 250 ul lysis buffer (50 mM Tris-HCl with 1 ug/ml lysozyme) andincubated on ice for 30 min. For fecal samples, one pellet was weighedand homogenized in 1 ml lysis buffer. Bacterial and fecal suspensionswere then sonicated (QSonica 125) and centrifuged at 12000×g for 10 minat 4° C. 20 ul supernatant was mixed with 180 ul substrate buffer (2.9mM L-gamma-glutamyl-3-carboxy-4-nitroanilide (Gold Bio), 100 mM ofglycylglycine (Sigma Aldrich), 100 mM Tris-HCl), and 500 uM GGsTop (ifnoted). Absorbance at 405 nm denoting production of3-carboxy-4-nitroaniline was measured every minute for 1 hr at 37° C.using an automated multimode plate reader (Biotek Synergy H1).

Intestinal Permeability Assay

Mice were fasted for 4 hr beginning at 7 am before gavage with 0.6 g/kg4 kDa FITC-dextran (Sigma Aldrich). 4 hours after gavage, serum sampleswere collected by cardiac puncture, diluted 3-fold in water and read induplicate for fluorescence intensity at 521 nm using a Synergy H9multimode plate reader (Biotek) against a standard dilution series ofstock FITC-dextran in 3-fold diluted normal mouse serum in water.

Statistical Analysis

Statistical analysis was performed using Prism software (GraphPad). Datawere assessed for normal distribution and plotted in the figures asmean±s.e.m. For each figure, n=the number of independent biologicalreplicates. No samples or animals were excluded from the analyses.Differences between two treatment groups were assessed using two-tailed,unpaired Student t test with Welch's correction. Differences among 22 2groups with only one variable were assessed using one-way ANOVA withBonferroni post hoc test. Data for Kcnl mice were analyzed bynon-parametric one-way ANOVA with Dunn's post hoc test. Two-way ANOVAwith Bonferroni post-hoc test was used for ≥2 groups with two variables(e.g. seizure time course, BHB time course, metabolomics data, bacterialgrowth curves). One-way ANOVA with repeated measures and Bonferronipost-hoc test was used for GGT assays. Significant differences emergingfrom the above tests are indicated in the figures by *P<0.05, **P<0.01,***P<0.001, ****P<0.0001 Notable near-significant differences(0.5<P<0.1) are indicated in the figures. Notable non-significant (andnon-near significant) differences are indicated in the figures by“n.s.”.

Example 2 Ketogenic Diet Alters Gut Microbiota and Confers Protectionagainst Seizures

The 6-Hz psychomotor seizure model of refractory epilepsy involveslow-frequency corneal stimulation to induce focal dyscognitive seizuresreminiscent of human temporal lobe epilepsy. The KD protects against6-Hz seizures, as indicated by the increased current intensity requiredto elicit a seizure in 50% of the subjects tested (CC50, seizurethreshold). Specific pathogen-free (SPF) Swiss Webster mice were fed a6:1 fat:protein KD or a vitamin- and mineral-matched control diet (CD).Compared to CD controls, mice consuming KD exhibited elevated seizurethresholds in response to 6-Hz stimulation (FIG. 1A), decreased serumglucose (FIG. 1B) and increased serum β-hydroxybutyrate (BHB; FIG. 1C).There were no significant differences in food consumption or weight gainacross CD vs. KD groups.

In addition to raising seizure thresholds, the ketogenic diet alteredthe composition of the gut microbiota (FIG. 1D), decreased a-diversity(FIG. 1E) and increased relative abundance of Akkermansia muciniphila(FIG. 1F). Parabacteroides, Sutterella and Erysipelotrichaceae spp. werealso elevated in KD-fed mice, whereas Allobaculum, Bifidobacterium andDesulfovibrio spp. were elevated in mice fed the control diet (FIG. 2).These results revealed that the composition of the gut microbiota wasrapidly and significantly altered in response to the KD.

Example 3 Gut Microbiota Confer the Anti-Seizure Effects of theKetogenic Diet

The 6-Hz psychomotor seizure threshold for germ-free (GF) and antibiotic(Abx)-treated SPF mice was measured in order to determine if the gutmicrobiota was necessary for the anti-seizure effects of the ketogenicdiet.

Compared to CD controls (SPF CD), SPF mice fed the KD (SPF KD) for 14days exhibited increased seizure thresholds and altered microbiota (FIG.3A and FIG. 1D). This was abrogated in GF mice (FIG. 3A) and Abx-treatedSPF mice (FIG. 3C), indicating that the gut microbiota was required forKD-mediated increases in seizure protection. Conventionalization of GFmice with the SPF gut microbiota restored KD-associated seizureprotection to levels seen in native SPF KD mice (FIG. 3A), whichsuggested that microbial mediation of KD seizure resistance was notdependent on pre-weaning microbiota colonization and that the microbiotaactively mediated seizure protection by the KD. Notably, microbialmodulation of KD-related seizure resistance did not correlate withchanges in serum BHB or glucose levels (FIG. 3B and FIG. 3D). There werealso no significant differences between groups in levels of intestinal,liver, or brain BHB. Overall, these data demonstrated that the gutmicrobiota was required for anti-seizure effects of the KD in the 6-Hzseizure model and further suggested that gut microbes modulated seizuresusceptibility through mechanisms that do not involve alterations in BHBlevels.

To determine whether specific bacterial taxa mediated seizure protectionin response to the KD, Abx-treated SPF mice were colonized with selectKD-associated bacteria, fed the KD and then tested for 6-Hz seizures(FIG. 4). Mice were gavaged with 10⁹ cfu bacteria: i) A. muciniphila,ii) 1:1 ratio of Parabacteroides merdae and P. distasonis, asrepresentative intestinal Parabacteroides spp. from the human microbiotawith highest homology to the Parabacteroides operational taxonomic unitreads that were enriched by the KD (FIG. 1D); or iii) 2:1:1 ratio of A.muciniphila, P. merdae and P. distasonis. At 14 days after gavage, 16SrDNA sequencing of colonic lumenal contents revealed that mice treatedwith A. muciniphila harbored 43.7±0.4% relative abundance of A.muciniphila. Mice gavaged with Parabacteroides spp. harbored 70.9±4.0%relative abundance, and mice gavaged with both taxa harbored 49.0±4.1%A. muciniphila and 22.5±5.4% Parabacteroides. Consistent with this, FISHprocessing of colonic sections from mice treated with A. muciniphila andParabacteroides spp. exhibited increased hybridization of the A.muciniphila probe MUC1437 and the Bacteroides and Parabacteroides spp.probe BAC303, where the average distance from a BAC303-positive cell tothe nearest MUC1437-positive cell is 0.64±0.09 microns. Both A.muciniphila and Parabacteroides spp. localized to the lumen of the mousecolon, not the mucosal space. There were no significant differences inweight, serum glucose levels, or enrichment of A. muciniphila andParabacteroides spp. across mice fed CD vs. KD. BHB was similarlyinduced in KD-fed groups, independent of colonization and seizurestatus. These data revealed that microbiota depletion by Abx treatmentfollowed by oral gavage of exogenous bacteria resulted in theirpersistent intestinal enrichment by 14 days post-inoculation.

Treatment with the KD alone elevated seizure thresholds by 24.5% from19.4±0.8 mA, in SPF CD mice, to 24.2±0.3 mA, in SPF KD mice. Whileco-administration of A. muciniphila and Parabacteroides spp. restoredprotection against 6-Hz seizures in Abx-treated mice fed the KD, raisingthresholds by 36.0%, from 19.9±0.3 mA, in Abx KD mice, to 27.0±0.5 mA,in AkkPb KD mice (FIG. 4A). The seizure protective effect of bacterialenrichment was specific to A. muciniphila with P. merdae, as micegavaged with A. muciniphila and P. distasonis exhibited no restorationof seizure protection. There was no significant increase in seizurethreshold after enrichment of either A. muciniphila or Parabacteroidesspp. alone (FIG. 4A), indicating that both were required for mediatingthe anti-seizure effects of the ketogenic diet. There also was no effectof treatment with Bifidobacterium longum (FIG. 4A), which was increasedin CD-treated mice (FIG. 1F). Moreover, co-colonization of A.muciniphila and Parabacteroides spp. in GF mice promoted seizureprotection in response to the KD, when compared toParabacteroides-monocolonized or A. muciniphila-monocolonized in GF mice(FIG. 4C). Overall, these findings revealed that A. muciniphila andParabacteroides spp. increased in response to the ketogenic diet andmediated its protective effect in the 6-Hz seizure model.

Example 4 The Gut Microbiota Sufficiently Confers Seizure Protection toMice Fed the Control Diet

To determine whether KD-associated gut microbes also conferredanti-seizure effects to mice fed the control diet, Abx-treated mice weretransplanted with CD vs. KD microbiota from SPF mice, fed the CD or KDand tested for their susceptibility to 6-Hz seizures after 4 days ofdietary treatment. Abx-treated mice were used to mimic clinical fecaltransplant approaches that involve pre-treatment with Abx to deplete thenative microbiota. Day 4 was selected based on i) the ability of the KDto induce significant microbiota changes by that time (FIG. 1D, FIG. 1F,and FIG. 5A) and ii) evidence that the KD microbiota exhibits incompletereversion to CD profiles at 4 days after switching from KD to CD (FIG.6A). Mice transplanted with a CD microbiota and fed KD for 4 daysdisplayed increased seizure threshold compared to CD-fed controls (FIG.5A). Abx-treated mice transplanted with a KD microbiome but fed CD for 4days exhibited seizure protection as well. This suggested thatcolonization with the KD microbiota raised seizure thresholds in micefed CD. Notably, however, seizure protection was abrogated aftercomplete reversion of the KD microbiota to CD profiles on day 28 (FIG.6B), which suggested that persistent interactions between the KDmicrobiota, diet and neuronal activity were required. Similaranti-seizure effects were seen after enriching A. muciniphila andParabacteroides spp. in Abx-treated SPF mice fed CD as compared toParabacteroides spp., A. muciniphila or B. longum controls (FIG. 5B).However, increases in seizure threshold in SPF CD mice treated with Abxalone relative to SPF CD controls confounded interpretation of theseresults (FIG. 5B). To clarify this uncertainty, a bacterial treatmentapproach to investigate whether exogenous treatment with A. muciniphilaand Parabacteroides spp. conferred anti-seizure effects in mice fed CDwas applied. SPF CD mice were gavaged bi-daily for 28 days with 10⁹ cfuA. muciniphila and Parabacteroides spp. or with vehicle. This bacterialtreatment increased seizure thresholds relative to vehicle-gavagedcontrols (FIG. 6C). Consistent with experiments on mice fed theketogenic diet (FIG. 4), this seizure protection was not observed inanimals treated with A. muciniphila alone, which revealed thatco-administration of A. muciniphila and Parabacteroides spp. wasrequired for seizure protection (FIG. 5C). Moreover, treatment withheat-killed bacteria decreased seizure thresholds compared tovehicle-treated controls, which suggested that viable bacteria werenecessary for conferring anti-seizure effects and release of bacterialcell surface and/or intracellular factors promoted sensitivity to 6-Hzseizures. Persistent exposure to A. muciniphila and Parabacteroides spp.was required, as increases in seizure thresholds were lost after ceasingtreatment for 21 days (FIG. 6C). In addition, seizure protection was notobserved in mice treated for only 4 days (FIG. 6D), which suggested thatlong-term exposure was required. Taken together, these findings revealedthat fecal transplant of the KD microbiota and bacterial treatment withthe KD-associated taxa A. muciniphila and Parabacteroides spp. conferredprotection against 6-Hz psychomotor seizures in mice fed the controldiet.

Example 5 KD Associated Bacteria Reduced Tonic-Clonic Seizures inKenal^(−/−) Mice

Epilepsy is a heterogeneous disorder with diverse clinicalpresentations. To determine whether the microbiota affected differentseizure types, the roles for the gut microbiota in modulatinggeneralized tonic-clonic seizures were tested in the Kcnal^(−/−) mousemodel for temporal lobe epilepsy and sudden unexpected death in epilepsy(SUDEP). Kcnal^(31 /−) mice harbor a null mutation in the voltage-gatedpotassium channel Kv1.1 alpha subunit, mimicking associations of humanKCNAL gene variants with epilepsy, episodic ataxia and SUDEP.Kcnal^(31 /−) mice develop severe spontaneous recurrent seizures, whichare reduced 54% by the KD. Kcnal^(−/−) SPF C3HeB/FeJ mice were treatedwith Abx or vehicle for 1 week, gavaged with vehicle or A. muciniphilaand Parabacteroides spp., and fed KD or CD for 3 weeks. Seizurefrequency and duration were recorded by EEG over 3 days, whereelectrographic seizures were identified based on characteristicepileptiform spike patterns consisting of 5 phases (FIG. 7C): A)low-frequency background, with low-voltage spiking, B) synchronizedhigh-frequency, high-voltage spiking, C) high-frequency, low-voltagespiking, D) unsynchronized high-frequency, high-voltage spiking, and E)high-frequency, burst spiking. Furthermore, EEG seizure patterns werecorroborated with stereotyped seizure behaviors identified by 5 stages.There were no significant differences in weight gain, food consumptionbetween mice fed KD vs. CD. No differences in survival across groupswere observed. Compared to CD-fed Kcnal^(−/−) controls, KD-fedkcnal^(−/−) mice exhibited altered gut microbiota profiles (FIG. 7A),with increases in A. muciniphila and Parabacteroides spp. Notably, thesechanges were mild and not statistically significant compared to theKD-induced enrichment seen in Swiss Webster mice (FIG. 1F), whichhighlighted an effect of host genotype on baseline microbiotacomposition and responses to KD. Vehicle-treated Kcnal^(−/−) miceexhibited seizures that lasted 15-180 seconds, with an average maximumspike amplitude of 490±26 uV (FIG. 7C). Decreases in seizure incidenceand duration in KD-fed Kcnal^(−/−) mice compared to CD-fed Kcnal^(−/−)controls were observed (FIG. 7D), consistent with KD-mediated seizureprotection as previously described. Kcnal^(−/−) mice that werepre-treated with Abx to deplete the gut microbiota exhibited asignificant increase in seizures per day and total seizure durationcompared to vehicle-treated, KD-fed Kcnal^(−/−) controls (FIG. 7D).There was no significant difference in spike frequency, interspikeinterval, and average duration per seizure which suggested a primaryeffect of Abx treatment and depletion of the gut microbiota on seizureoccurrence. Moreover, colonization of Abx-treated Kcnal^(−/−) mice withA. muciniphila and Parabacteroides spp. reduced seizure frequency andtotal duration of seizures toward levels seen in vehicle-treated, KD-fedKcnal^(−/−) controls (FIG. 7D). This suggested that treatment with A.muciniphila and Parabacteroides spp. similarly conferred seizureprotection in mouse strains that have different baseline anddiet-altered microbiota (in this case, C57B1/6 vs. C3HeB/FeJ). Takentogether, these findings supported the notion that select bacterialspecies from the indigenous gut microbiota mediated the anti-seizureeffects of the KD across varied seizure types and models.

Example 6 Microbiota Modulated Gut, Serum, and Brain Metabolomes

Metabolomic profiling was used to identify candidatemicrobiota-dependent molecules in colonic lumenal contents and sera ofSPF mice fed CD, and SPF, Abx-treated SPF, and A. muciniphila- andParabacteroides spp.-enriched mice fed KD (FIG. 8A and FIG. 9A).Metabolomic profiles in colonic lumenal contents and sera discriminatedseizure-protected (vehicle-treated SPF mice fed KD and A. muciniphilawith Parabacteroides spp.—enriched mice fed KD) from seizure-susceptible(vehicle-treated SPF mice fed CD and Abx-treated SPF mice fed KD)groups, with a predictive accuracy of 94% for colonic lumenalmetabolites and 87.5% for serum metabolites. The majority of metabolitesthat contributed highly to group discrimination were relevant to aminoacid metabolism, including derivatives of lysine, tyrosine andthreonine. In addition, widespread decreases in subsets of ketogenicgamma-glutamylated amino acids—gamma-glutamyl (GG)-leucine, GG-lysine,GG-threonine, GG-tryptophan and GG-tyrosine—in colonic lumenal contents(FIG. 8C) and sera (FIG. 8D) from seizure-protected compared toseizure-susceptible groups was observed. This suggested that the gutmicrobiota modulated gamma-glutamylation itself or selective metabolismof ketogenic GG-amino acids and that increased ketogenic GG-amino acidswere associated with seizure susceptibility. Supporting this notion,imputed metagenomes predicted KD-associated alterations in bacterialgenes relevant to amino acid metabolism. These data revealed significanteffects of the gut microbiota on intestinal and systemic metabolomicresponses to the KD, and further revealed an association betweenKD-induced seizure protection and microbiota-dependent alterations inlevels of ketogenic GG-amino acids.

The brain relies on active import of essential amino acids to fuelneurotransmitter biosynthesis, and as such, is sensitive to fluctuationsin peripheral amino acid bioavailability. GG-amino acids, in particular,are hypothesized to exhibit increased transport properties compared tonon-gamma-glutamylated forms. Based on data that revealed diet- andmicrobiota-dependent alterations in serum ketogenic amino acids, linksbetween amino acids importation and brain GABA levels, and prevailingtheories that GABA contributed to the anti-seizure effects of the KD,bulk levels of GABA and glutamate in the hippocampus, a key region forseizure propagation, were examined. Hippocampal metabolite profilesdistinguished samples for seizure-protected vs. seizure-susceptiblemice. Hippocampal GABA/glutamate ratios are significantly increased inKD-fed SPF mice compared to CD-fed controls (FIG. 8D, left). Theseincreases were abrogated in Abx-treated mice fed KD and restored afterenrichment of Abx-treated mice with A. muciniphila and Parabacteroidesspp. (FIG. 8D). Similar changes were seen for hippocampal levels ofglutamine, a precursor of glutamate and GABA (FIG. 8D, right). Overall,these results revealed diet- and microbiota-dependent regulation in thebioavailability of glutamine, as well as a preferential increase inhippocampal GABA levels relative to glutamate in seizure-protected mice.

Example 7 Bacterial Gamma-Glutamylation Impacted Seizure Susceptibility

Based on the finding that essential ketogenic GG-amino acids werereduced in colonic lumen and serum of seizure-protected vs.seizure-susceptible experimental groups, it was hypothesized thatmicrobiota-dependent restriction of ketogenic GG-amino acids isimportant for mediating the anti-seizure effects of the KD.Gamma-glutamylated forms of amino acids were generated bytranspeptidation of GG moieties from glutathione onto amino acids. Todetermine whether gamma-glutamylation of amino acids impacts seizuresusceptibility, SPF CD mice were gavaged for 3 days with GGsTop, aselective irreversible inhibitor of GGT. SPF CD mice treated with GGsTopexhibited increases in seizure thresholds toward levels seen in SPF KDmice (FIG. 10A). Similarly, EEG recordings of CD-fed SPF Kcnal−/− micetreated with GGsTop displayed a significant decrease in seizures per day(FIG. 7E). This demonstrated that peripheral inhibition ofgamma-glutamylation and restriction of GG-amino acids promoted seizureprotection, consistent with observed metabolomic decreases of ketogenicGG-amino acids in colonic lumenal content and sera fromseizure-protected groups compared to seizure-susceptible controls. Todetermine whether restriction of amino acids, rather than catabolism ofglutathione, was necessary for the anti-seizure effects of the KDmicrobiota, KD-fed A. muciniphila and Parabacteroides spp.-enriched micewere supplemented by bi-daily intraperitoneal injection for 3 days withcombined leucine, lysine, threonine, tryptophan and tyrosine, and thentested for 6-Hz seizures. Physiologically-relevant concentrations ofamino acids were calculated based on serum metabolomic data, such thatdosages for each restored blood levels to that seen in vehicle-treatedSPF CD controls. Elevating systemic levels of ketogenic amino acidsdecreased seizure thresholds to levels seen in vehicle-treated SPF CDcontrols (FIG. 10B). This suggested that restriction of peripheralketogenic amino acids was necessary for mediating microbiota- andKD-dependent increases in seizure resistance.

Both host cells and particular bacterial species exhibit GGT activity.To gain insight into whether the KD and interactions between A.muciniphila and Parabacteroides spp. suppressed bacterialgamma-glutamylation in vivo, GGT activity was measured in fecal samplescollected from SPF or A. muciniphila and Parabacteroides spp.-enrichedmice fed the CD or KD. Feeding SPF mice with KD decreased fecal GGTactivity compared to CD controls (FIG. 10C). Similar reduction in fecalGGT activity was seen after enriching A. muciniphila and Parabacteroidesspp. in CD-fed mice. Moreover, enriching A. muciniphila andParabacteroides spp. and feeding with KD further decreased fecal GGTactivity relative to that seen in SPF KD and SPF CD mice. Exposing allfecal samples to the GGT inhibitor GGsTop eliminated the detectedsignals, confirming that the measurements reflected GGT activity.Consistent with this, treatment of CD-fed SPF mice with A. muciniphilaand Parabacteroides spp. decreased fecal GGT activity relative tovehicle-treated controls and mice treated with heat-killed bacteria(FIG. 10D). Overall, these data revealed that enriching for or exogenoustreatment with A. muciniphila and Parabacteroides spp. reduced fecal GGTactivity, which could explain the low levels of colonic and serumGG-amino acids observed in seizure-protected mice.

To explore whether bacterial gamma-glutamylation was affected byinteractions between A. muciniphila and Parabacteroides spp., GGTactivity in bacteria grown in an in vitro cross-feeding system wasmeasured. When A. muciniphila was embedded in a CD- or KD-based agar,and P. merdae was overlaid in M9 minimal media over the agar, bothbacteria exhibited enhanced growth (FIG. 10E and FIG. 10F), whichsuggested that A. muciniphila liberated soluble factors to enable P.merdae growth and in turn P. merdae enhanced A. muciniphila growth.Pilot experiments revealed no growth of A. muciniphila in M9 media whenoverlaid on P. merdae embedded in KD or CD agar, which suggested that A.muciniphila cannot rely solely on cross-feeding from P. merdae topersist.

P. merdae exhibited high GGT activity that was eliminated by theaddition of A. muciniphila embedded in CD or KD agar (FIG. 10G and FIG.10H). To determine whether reduction of GGT activity in P. merdaepromoted A. muciniphila growth, P. merdae was pre-treated with vehicleor GGsTop to pharmacologically inhibit GGT activity prior to testing inthe cross-feeding assay. A. muciniphila exposed to P. merdae that waspre-treated with GGsTop exhibited increased growth at 24 hours afterincubation as compared to A. muciniphila exposed to vehicle-treated P.merdae (FIG. 11B). Taken together, these findings suggested that A.muciniphila was capable of metabolizing components from the KD and CDdiet to support P. merdae growth, and that the cooperative interactionreduced GGT activity. In turn, reductions in GGT activity in P. merdaepromotes A. muciniphila growth. This was consistent with the findingthat enrichment of A. muciniphila and Parabacteroides spp. reduced fecalGGT activity, colonic lumenal GG-amino acids and serum GG-amino acids.This demonstrated that amino acid restriction was required for seizureprotection and that inhibition of GGT promoted seizure protection. Thisaligned with previous studies linking GGT activity to altered seizureseverity. In a study of 75 epileptic patients, high serum GGT activitywas observed in 84.5% of the patients compared to controls. In a ratseizure model, GGT activity was increased after 5 consecutive dailyelectroshock deliveries. Decreases in various peripheral amino acids areassociated with KD-mediated seizure suppression in animals and humans.

Based on the data herein and existing literature on roles for peripheralamino acids as substrates for brain neurotransmitter biosynthesis, itwas hypothesized that bacterial regulation of GG-amino acids alteredbrain import of amino acids that fuel GABA/glutamate metabolism (FIG.8). Notably, several gut bacteria are reported to synthesize GABA denovo; however, circulating GABA exhibits limited transport across theblood-brain barrier. In addition, changes in the gut microbiota havebeen associated with alterations in brain GABA levels, but the molecularmechanisms involved remain unclear. Additional studies are needed todetermine whether GG-amino acids influence brain transport of aminoacids and local synthesis of glutamate versus GABA.

Overall, this study demonstrated a novel role for select KD-associatedgut bacteria—A. muciniphila and Parabacteroides spp.—in mediating andconferring seizure protection in mouse models for refractory epilepsy.Increases in A. muciniphila were similarly observed during fasting inhumans, hamsters, squirrels, and pythons, and in response to caloricrestriction and high polyunsaturated fat diets in mice. A. muciniphilaand Parabacteroides spp. are also positively associated with increasedketosis and the ketogenic diet in humans. The data herein reveals alikely pathway whereby the KD promoted select microbe-microbeinteractions that reduced host levels of ketogenic GG-amino acids andelevated the total bioavailability of GABA relative to glutamate in thehippocampus. Pharmacological inhibition of gamma-glutamylation increasedseizure thresholds, which suggested that reduced GGT activity wasimportant for mediating the anti-seizure effects of the KD-associatedgut microbiota in mice. Notably, given that lack of bacterial GGTactivity in the GF condition was associated with seizure susceptibility,it is likely that A. muciniphila and Parabacteroides spp. contributedfunctions in addition to suppression of GGT activity that may alsocontribute to seizure protection.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. A method of preventing or treating a condition responsive to aketogenic diet in a subject, comprising administering to the subject acomposition comprising bacteria of the Akkermansia (Akk) andParabacteroides (Pb) genera.
 2. The method of claim 1, wherein themethod prevents or treats the condition by altering neurotransmitterbiosynthesis, by altering serum ketogenic amino acids, by decreasinggamma-glutamyltranspeptidase activity, by decreasing glutamine synthaseactivity, by decreasing gamma-glutamyl amino acids, by increasingGABA/glutamate ratio levels, by increasing glutamine levels, or by acombination thereof in the subject. 3-8. (canceled)
 9. The method ofclaim 1, wherein the bacteria of the Akkermansia (Akk) genus compriseAkkermansia muciniphila.
 10. The method of claim 1, wherein the bacteriaof the Parabacteroides (Pb) genus comprise Parabacteroides merdae,Parabacteroides distasonis, or both Parabacteroides merdae andParabacteroides distasonis.
 11. (canceled)
 12. The method of claim 1,wherein at least 10%, at least 30%, at least 50%, at least 70%, or atleast 90% of the bacteria in the composition are Akkermansia (Akk).13-16. (canceled)
 17. The method of claim 1, wherein at least 10%, atleast 30%, at least 50%, at least 70%, or at least 90% of the bacteriain the composition are Parabacteroides (Pb) bacteria. 18-21. (canceled)22. The method of claim 1, wherein the condition is selected fromseizures, Alzheimer's disease, Huntington's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), cancer, stroke, ametabolic disease, a mitochondrial disorder, depression, migraines,traumatic brain injury (TBI), epilepsy, autism spectrum disorder, Rettsyndrome, attention deficit disorder, and fragile X syndrome. 23.(canceled)
 24. The method of claim 1, wherein the subject is on a diet,and the diet is a control diet, a ketogenic diet, a high fat diet, or alow carbohydrate diet. 25-27. (canceled)
 28. The method of claim 1,wherein the composition is formulated for oral delivery or for rectaldelivery.
 29. The method of claim 1, wherein the composition is a foodproduct.
 30. The method of claim 29, wherein the food product is a dairyproduct or yogurt. 31-32. (canceled)
 33. The method of claim 1, whereinthe composition is self-administered.
 34. The method of claim 1, furthercomprising subject. 35-48. (canceled)
 49. A composition comprisingbacteria of Akkermansia (Akk) and Parabacteroides (Pb) genera.
 50. Thecomposition of claim 49, wherein the bacteria of the Akkermansia (Akk)genus comprise Akkermansia muciniphila.
 51. The composition of claim 49,wherein the bacteria of the Parabacteroides (Pb) genus compriseParabacteroides merdae, Parabacteroides distasonis, or bothParabacteroides merdae and Parabacteroides distasonis.
 52. (canceled)53. The composition of claim 49, wherein at least 10%, at least 30%, atleast 50%, at least 70%, or at least 90% of the bacteria in thecomposition are Akkermansia (Akk) bacteria. 54-57. (canceled)
 58. Thecomposition of claim 49, wherein at least 10%, at least 30%, at least50%, at least 70%, or at least 90% of the bacteria in the compositionare Parabacteroides (Pb) bacteria. 59-62. (canceled)
 63. The compositionof claim 49, wherein the composition is formulated for oral delivery orfor rectal delivery.
 64. The composition of claim 63, wherein thecomposition is a food product.
 65. The composition of claim 64, whereinthe food product is a dairy product or yogurt. 66-92. (canceled)